D-Allulose is the C-3 epimer of D-fructose and is a low-caloric sweetener. Allulose, also widely known as D-psicose, is very similar to glucose in regards to intensity and sweetness. However, because the body metabolizes allulose differently than most sugars, such as glucose and fructose, its caloric value is significantly lower. In fact, its caloric value is nearly zero. Like glucose, D-allulose has about 70% of the relative sweetness of sucrose but only provides 0.2 kcal/mol energy.
The bio-conversion of D-fructose to D-allulose by D-tagatose-3-epimerase (DT3E) or by D-psicose-3-epimerase (FIG. 1) has long been recognized, however, different enzymes having the required activity have different properties such as pH and cofactor requirements, equilibrium constants, temperature tolerance and the like. For commercial production of D-allulose it is desirable to discover or engineer enzymes with robust and advantageous properties. The conversion of D-fructose to D-allulose will diversify the traditional sweetener product portfolio associated with corn processing by adding a natural low caloric sweetener and bulking agent to the traditional portfolio of sweeteners derived from corn starch, i.e. corn syrup, high fructose corn syrup (HFCS), glucose and fructose.
Most of the epimerases that have been identified to date are of bacterial origin being principally derived from soil bacteria exemplified by Pseudomonas sp., Agrobacterium sp., Rhizobium sp., Clostridium sp., Desmospora sp., Rhodobactor sp., and Arthobactor sp. Most of these epimerases show dependence on manganese and/or cobalt as a cofactor and are inactive in absence of these metals. Notable exceptions are the epimerase from P. chicorii and A. globiformis which show activity in the presence of Mg+2. The use of Mg+2 as a metal cofactor instead of Mn+2 or Co+2 provides a significant advantage when deploying these enzymes in commercial production, which helps in process integration with existing fructose production operations and avoids issues related to waste water treatment.
FIG. 2 is a table that list various properties for several known epimerases suggested for use in allulose production. The optimal pH range for these epimerases is between 7.0 and 9.0 with the majority being between 7.0 and 8.0. The optimum temperature ranges between 40° C. and 70° C. with the great majority being in the range of 55-60° C. In order to have the best catalytic efficiency the reaction should be operated as close to the optimum pH and temperature as is practical. For commercial production, it is desirable to use higher temperatures of 60° C. or greater which allow a higher dissolved solids content for the input and output streams. However, fructose and allulose are subject to degradation at optimal operational pH's and temperatures. At a temperature of 60° C. fructose and allulose stability is best between pH 4.5 and 5.5. Operating the process at a pH of 7-8 and at such high temperatures results in formation of byproducts in the reaction mixture that leads to yield loss and requires removal of color bodies from the final product.
Therefore, there is a need in the art to discover new classes of epimerases that can convert fructose to allulose at low pH and high temperatures which can do so at a high dissolved solids content using Mg+2 as a metal cofactor. There is also a need to provide recombinant DNA expression systems for efficient expression of such epimerases from bacterial sources.